Determination of green tea catechins in human plasma using liquid chromatography-electrospray ionization mass spectrometry.

A method for the sensitive and specific determination of eight green tea catechins, consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin-3-gallate (CG), epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and epigallocatechin-3-gallate (EGCG), in human plasma was established. For optimization of conditions for LC-ESIMS, the separation of the eight catechins was achieved chromatographically using Inertsil ODS-2 column combined with a gradient elution system of 0.1M aqueous acetic acid and 0.1M acetic acid in acetonitrile. Detection using a mass spectrometer was performed with selected ion monitoring at m/z=289 for E and EC, 305 for GC and EGC, 441 for CG and ECG, and 457 for GCG and EGCG under negative ESI. A preparative procedure, consisting of the addition of perchloric acid and acetonitrile to the plasma for deproteinizing and the subsequent addition of potassium carbonate solution to remove excess acid, was developed. In six different plasma with the eight catechins spiked at two different concentrations, the average recoveries were in the range between 72.7 and 84.1%, which resulted from the matrix effect and preparative loss, with coefficients of variance being 8.2-19.8% among individuals. The levels of the catechins in prepared plasma solutions that were kept at 5 degrees C within 24h were stable, which allows us to simply analyze many prepared plasma solutions using an autosampler overnight. When using this method to analyze the eight catechins in human plasma after oral ingestion of a commercial green tea beverage, we detected all the catechins absorbed into human blood for the first time. This also suggested that extremely small amounts of the eight catechins orally ingested may be absorbed based on each absorptive property for the catechins. The method should enable pharmacokinetic studies of green tea catechins in humans.

Deficient production of hexadecenoic acid in the skin is associated in part with the vulnerability of atopic dermatitis patients to colonization by Staphylococcus aureus.

BACKGROUND AND OBJECTIVES: As one of the major skin fatty acids, cis-6-hexadecenoic acid (C16:1Delta6) exhibits a specific antibacterial activity and might play a specific role in the defense mechanism against Staphylococcus aureus, in healthy subjects whereas S. aureus frequently colonizes the skin of patients with atopic dermatitis (AD). METHODS: Fatty acid composition of sebum at the recovery level was analyzed by gas chromatography and S. aureus colonizing the skin was assessed by the 'cup-scrub' method (9 patients and 10 healthy controls). To evaluate in vivo effect of C16:1Delta6 on colonization, C16:1Delta6 was applied for 2 weeks on the upper arm skin of another group of AD patients (11 patients). RESULTS: Analysis of sebum lipids revealed that there is a significant lower free C16:1Delta6 content in nonlesional skin from AD patients compared with healthy controls. This lower content is also accompanied by a significantly lower level of C16:1Delta6 in the total fatty acid composition of sebum (analyzed following hydrolysis). The lower level of free C16:1Delta6 correlated significantly (R(2) = 0.41, p < 0.01) with the numbers of S. aureus colonizing nonlesional skin. Topical application of free C16:1Delta6 on the skin of AD patients for 2 weeks abolished the markedly increased bacterial count in 6 out of the 8 AD patients tested. CONCLUSIONS: Free C16:1Delta6 may be involved in the defense mechanism against S. aureus in healthy skin and this deficit triggers the susceptibility of the skin to colonization by S. aureus in AD.